The present invention relates to novel substituted pyridine compounds having estrogenic activity, to processes for their preparation, to combinatorial and solid phase methods for preparing libraries of the compounds, to utilizing libraries of the compounds for drug discovery, to methods of treatment and to pharmaceutical :compositions thereof.
The solid phase synthesis of non-peptidic small organic molecules is a rapidly evolving area of research with applications in the preparation of combinatorial libraries. While the solid phase synthesis of peptides is an established, the solid phase synthesis of non-peptidic small organic molecules is still evolving (Hermkens, P. H. H.; Ottenheijm, H. C. J.; Rees, D. Tetrahedron 1996, 52, 4527-4554). In particular, methods for the solid phase synthesis of heterocyclic ring systems of importance to drug discovery is an active area of research.
Pyridine derivatives are commonly used as pharmaceuticals (Gordeev, M. F., et al. Tetrahedron Lett., 1996, 37, 4643-4646). Trisubstituted pyridines are a useful class of compounds. Karle, et al. (Antimicrob. Agents Chemother. 1989, 33, 1081-1089) describe 2,4,6-trisubstituted pyridines as antiprotozoal agents. Shirai, et al. (WO 96/00213) describe 2,4,6-trisubstituted pyridines as useful for accelerating nerve growth factor production, and also ((WO 96/16942) as useful for ameliorating neuropathy.
Combinatorial chemistry is becoming an important tool for drug discovery and lead optimization (Borman, S. Chemical and Engineering News 1997, 75 (8), 43-62). A combinatorial synthesis requires that at least two components of the product molecules be independently variable, so that all of the combinations of these components can be prepared. A synthesis with three independently variable components is preferable since greater diversity in structure can be produced in the resultant library. Thus, to prepare a combinatorial library of pyridines with a high degree of potential diversity and wide utility for drug discovery using solid phase techniques, it is important to identify a pyridine synthesis in which three components can be independently varied. The solution phase synthesis of pyridines from 1,5-pentanediones and ammonia followed by oxidation is known (Katritzky, A. R. Handbook of Heterocyclic Chemistry, pp. 408-409; Pergamon Press: Oxford, 1985). A variation of this synthesis involves the reaction of a bromomethyl ketone with pyridine, and subsequent reaction of this intermediate with an unsaturated ketone in the presence of ammonium acetate in acetic acid to yield a 2,4,6-trisubstituted pyridine (Krohnke, F. Synthesis 1976, 1-24). The latter synthesis proceeds through a 1,5-diketone intermediate which is not isolated. For a solid phase combinatorial synthesis it is necessary to modify these syntheses to allow for the independent introduction of three variables (the 2,4, and 6 substituents), and to adapt the solution phase synthesis to a solid supported synthesis. The solid phase pyridine synthesis of this invention is achieved by using a hydroxyacetophenone starting material which can be attached to a solid support through the phenolic hydroxy group.
A solid phase synthesis of 2,3,4,5,6-pentasubstituted dihydropyridines and pyridines has been described in Gordeev, M. F., et al. Tetrahedron Lett., 1996, 37, 4643-4646. The compounds are prepared by the Hantzsch pyridine synthesis and therefore all contain acyl or carboxyl groups in the 3- and 5-positions. The solid phase synthesis of the current invention does not yield pyridines with acyl or carboxyl groups in the 3- and 5-position of all products and therefore yields a more diverse set of products.
Multiple compounds can be prepared simultaneously by the solid phase process. The simultaneous solid phase synthesis of a library of 2,4,6-trisubstituted pyridines of the present invention is not known. The preparation of libraries of compounds of the present invention is useful because it provides rapid structural variation and structure-activity information.
The libraries of substituted pyridines synthesized according to the present invention are useful for drug discovery. Screening of the pyridine libraries in an estrogen receptor assay identified compounds with estrogen agonist activity. Estrogen agonists are useful as post-menopausal therapeutics for the prevention and treatment of osteoporosis, atherosclerosis, and Alzheimer""s disease.
The present invention relates to new compounds selected from those of the general Formula (I) and also discloses a solid phase synthesis process for producing new compounds selected from those of Formula (I): 
wherein
the moiety Z 
is selected from the group: 
n is an integer of 1 or 2;
R1 is straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cycloalkyl of 3 to 7 carbon atoms, phenyl, or phenyl substituted with fluoro, chloro, bromo, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cycloalkyl of 3 to 7 carbon atoms, phenyl, alkoxy of 1 to 6 carbon atoms, or methylenedioxy;
R2 is furanyl, pyridyl, thienyl, naphthalenyl, phenyl, or phenyl substituted with fluoro, chloro, bromo, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cycloalkyl of 3 to 7 carbon atoms, alkoxy of 1 to 6 carbon atoms, or methylenedioxy;
R3 is hydrogen, fluoro, chloro, bromo, nitro, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cycloalkyl of 3 to 7 carbon atoms, or alkoxy of 1 to 6 carbon atoms;
and all crystalline forms and the pharmaceutically acceptable salts thereof, the enantiomers thereof, the racemic mixtures thereof, and the diastereomeric mixtures thereof.
Among the preferred groups of compounds of this invention are those in the subgroups:
a) compounds having the general formula: 
wherein R1, R2, and R3 are as defined above or a pharmaceutically acceptable salt;
b) compounds having the general formula: 
wherein R1, R2, R3 and n are as defined above or a pharmaceutically acceptable salt.
Among the more preferred compounds of this invention are those of the formula: 
wherein R1, R2, and R3 are as defined above or a pharmaceutically acceptable salt.
The most particularly preferred compounds of Formula (I) of the present invention prepared by the herein described solid phase synthesis processes are:
2-[6-(4-chloro-phenyl)-4-(3,4-difluoro-phenyl)-pyridin-2-yl]-phenol or a pharmaceutically acceptable salt thereof;
2-[4-(3,4-difluoro-phenyl)-6-naphthalen-2-yl-pyridin-2-yl]-phenol or a pharmaceutically acceptable salt thereof;
2-[4-(3,4-difluoro-phenyl)-6-furan-2-yl-pyridin-2-yl]-phenol or a pharmaceutically acceptable salt thereof;
2-(4-benzo[1,3]dioxol-5-yl-6-naphthalen-2-yl-pyridin-2-yl)-phenol or a pharmaceutically acceptable salt thereof;
2-(4-benzo[1,3]dioxol-5-yl-6-thiophen-3-yl-pyridin-2-yl)-phenol or a pharmaceutically acceptable salt thereof;
2-(4-biphenyl-4-yl-6-naphthalen-2-yl-pyridin-2-yl)-4-fluoro-phenol or a pharmaceutically acceptable salt thereof;
2-(4-biphenyl-4-yl-[2,4xe2x80x2]bipyridinyl-6-yl)-4-fluoro-phenol or a pharmaceutically acceptable salt thereof;
2-(4-cyclohexyl-6-furan-2-yl-pyridin-2-yl)-4-fluoro-phenol or a pharmaceutically acceptable salt thereof;
3-(4-biphenyl-4-yl-6-naphthalen-2-yl-pyridin-2-yl)-phenol or a pharmaceutically acceptable salt thereof;
3-(4-cyclohexyl-6-furan-2-yl-pyridin-2-yl)-phenol or a pharmaceutically acceptable salt thereof.
The novel process for producing novel compounds of Formula (I) comprises the steps of:
a) attaching a hydroxyacetophenone 1 of the formula 
or an alkaline metal salt thereof where the moiety Z and R3 are hereinbefore defined,to a solid support to produce an acetophenone 2 
wherein the moiety Z and R3 are hereinbefore defined, R4 and R5 are independently hydrogen or methoxy, and P is preferably a polystyrene resin support crosslinked with divinylbenzene;
b) reacting said acetophenone 2 with an aldehyde R1CHO wherein R1 is as hereinbefore defined, in the presence of a base to produce an olefin 3 
wherein the moiety Z, R1, R3, R4, R5, and P are as hereinbefore defined;
c) reacting olefin 3 with a silyl enol ether 4 
wherein R2 is as hereinbefore defined and TMS is trimethylsilyl, in the presence of a fluoride source such as cesium fluoride to produce 1,5-diketone 5 
wherein the moiety Z, R1, R2, R3, R4, R5, and P are as hereinbefore defined;
d) reacting 1,5-diketone 5 with ammonium acetate to produce pyridine 6 
wherein the moiety Z, R1, R2, R3, R4, R5 and P are as hereinbefore defined; and
e) reacting pyridine 6 with a cleaving reagent such as trifluoroacetic acid to produce a compound of Formula (I) 
wherein the moiety Z, R1, R2 and R3 are as hereinbefore defined.
The present invention also relates to new combinatorial compound libraries selected from those of the general Formula (I) and also discloses a solid phase synthesis process for producing new compound combinatorial libraries selected from those of Formula (I): 
wherein:
the moiety Z 
is selected from the group: 
n is an integer: of 1 or 2;
R1 is straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cycloalkyl of 3to 7 carbon aosphenyl, or phenyl substituted with fluoro, chloro, brormo, straight chain alkyl of 1 to 6 carbon atoms,. branched chain alkyl of 3 to 7 carbon atoms, cycloalkyl of 3 to 7 carbon atoms, phenyl, alkoxy of 1 to 6 carbon atoms, or methylenedioxy;
R2 is furanyl, pyridyl, thienyl, naphthalenyl, phenyl, or phenyl substituted with fluoro, chloro, bromo, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cycloalkyl of 3 to 7 carbon atoms, alkoxy of 1 to 6 carbon atoms, or methylenedioxy;
R3 is hydrogen, fluoro, chloro, bromo, nitro, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cycloalkyl of 3 to 7 carbon atoms, or alkoxy of 1 to 6 carbon atoms; and all crystalline forms and the pharmaceutically acceptable salts thereof, the enantiomers thereof, the racemic mixtures thereof, and the diastereomeric mixtures thereof.
Among the preferred combinatorial libraries of compounds of this invention are those in the subgroups:
a) combinatorial libraries of compounds having the general formula: 
wherein R1, R2, and R3 are as defined above or a pharmaceutically acceptable salt;
b) combinatorial libraries of compounds having the general formula: 
wherein R1, R2, R3 and n are as defined above or a pharmaceutically acceptable salt.
Among the more preferred combinatorial libraries of compounds of this invention are those of the formula: 
wherein R1, R2, and R3 are as defined above or a pharmaceutically acceptable salt.
It is understood that the definition of the compounds of Formula (I), when R1, R2, and R3 contain asymmetric carbons, encompasses all possible stereoisomers and mixtures thereof. In particular, it encompasses racemic modifications and any optical isomers. Optical isomers may be obtained in pure form by standard separation techniques. The pharmaceutically acceptable salts are those derived from such organic and inorganic acids as: lactic, citric, acetic, tartaric, succinic, maleic, malonic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, and similarly known acceptable acids. Carboxylic acid salts of the compounds of this invention may be formed with bases such as alkali metals (Na, K, Li) or the alkaline earth metals (Ca or Mg).
The compounds of the present invention may be prepared according to the general process outlined below in Scheme I. 
As shown in Scheme II a resin such as Wang resin 7 (R4 and R5=H, P=polystyrene crosslinked with divinylbenzene) (Wang S.; J. Am. Chem. Soc. 1973, 95, 1328-1333) is converted to a chloro resin 8 with lithium chloride, methanesulfonyl chloride, and a base such as collidine or lutidine in a polar aprotic solvent such as dimethylformamide. 
As outlined in Scheme I, chlororesin 8 is reacted with an alkaline metal salt, preferably the cesium salt, of a hydroxyacetophenone 1 where R3 is hereinbefore defined, to produce an acetophenone 2 where the moiety Z, R3, R4, R5 and P are hereinbefore defined. Acetophenone 2 is reacted with an aldehyde R1CHO where R1 is hereinbefore defined in the presence of a base such as sodium methoxide in a polar aprotic solvent such as trimethyl orthoformate at temperatures ranging from 0xc2x0 C. to 50xc2x0 C. to yield an olefin 3 on a solid support resin where the moiety Z, R1, R3, R4, R5 and P are hereinbefore defined. Olefin 3 is reacted with a silyl enol ether 4 where R2 is hereinbefore defined and TMS is trimethylsilyl in the presence of a fluoride source such as cesium fluoride in a polar aprotic solvent such as dimethyl sulfoxide at temperatures ranging from 25xc2x0 C. to 120xc2x0 C. to yield a 1,5-diketone 5 on a solid support where the moiety R1, R2, R3, R4, R5 and P are hereinbefore defined. A 1,5-diketone 5 is reacted with ammonium acetate in the presence of acetic acid in a polar aprotic solvent such as dimethylformamide at temperatures ranging from 25xc2x0 C. to 120xc2x0 C. to yield a pyridine 6 on a solid support where the moiety Z, R1, R2, R3, R4, R5 and P are hereinbefore defined. A compound of Formula (I) where the moiety Z, R1, R2 and R3 are as defined above is removed from the solid support with an acidic cleavage mixture such as trifluoroacetic acid in dichloromethane.
The present invention accordingly provides a pharmaceutical composition which comprises a compound of this invention in combination or association with a pharmaceutically acceptable carrier. In particular, the present invention provides a pharmaceutical composition which comprises an effective amount of a compound of this invention and a pharmaceutically acceptable carrier.
The compositions are preferably adapted for oral administration. However, they may be adapted for other modes of administration, for example, parenteral administration for patient suffering from heart failure.
It is understood that the dosage, regimen and mode of administration of these compounds will vary according to the malady and the individual being treated and will be subject to the judgement of the medical practitioner involved. It is preferred that the administration of one or more of the compounds herein begin at a low dose and be increased until the desired effects are achieved.
In order to obtain consistency of administration, it is preferred that a composition of the invention is in the form of a unit dose. Suitable unit dose forms include tablets, capsules and powders in sachets or vials. Such unit dose forms may contain from 0.1 to 100 mg of a compound of the invention and preferably from 2 to 50 mg. Still further preferred unit dosage forms contain 5 to 25 mg of a compound of the present invention. The compounds of the present invention can be administered orally at a dose range of about 0.01 to 100 mg/kg or preferably at a dose range of 0.1 to 10 mg/kg. Such compositions may be administered from 1 to 6 times a day, more usually from 1 to 4 times a day.
The compositions of the invention may be formulated with conventional excipients, such as a filler, a disintegrating agent, a binder, a lubricant, a flavoring agent and the like. They are formulated in conventional manner, for example, in a manner similar to that use for known antihypertensive agents, diuretics and beta-blocking agents.
The new compounds of Formula (I) of this invention are useful in treating conditions in mammals characterized by estrogen deficiency such as in post-menopausal women.
In particular, compounds of Formula (I) of this invention are useful as post-menopausal therapeutics for the prevention and treatment of osteoporosis, atherosclerosis, and Alzheimer""s disease in mammals.
The present invention further provides a compound of the invention for use as an active therapeutic substance.
Objective: To identify compounds that enhance the expression of luciferase gene activity compared to 17B-estradiol in a transient transfection model. Enhancement of luciferase gene expression in this model is dependent upon estrogen receptor (ER) interaction with a vitellogenin gene estrogen responsive element (ERE) capable of enhancing basal promoter activity. This is a sensitive and rapid methodology to assess estrogenic/antiestrogenic potency of compounds.
Procedure: Cell Maintenace and treatment: Chinese Hamster Ovary cells (CHO) which have been stably transfected with the human estrogen receptor are maintained in DMEM+10% fetal bovine serum (FBS). 48 h prior to treatment the growth medium is replaced with DMEM lacking phenol red+10% dextran coated charcoal stripped FBS (treatment medium). Cells are plated at a density of 5000 cells/well in 96-well plates containing 200 xcexcL of medium/well.
Calcium Phosphate Transfection: Reporter DNA (Promega plasmid pGL2 containing two tandem copies of the vitellogenin ERE in front of the minimal thymidine kinase promoter driving the luciferase gene) is combined with the B-galactosidase expression plasmid pCH110 (Pharmacia) and carrier DNA (pTZ18U) in the following ratio: 10 xcexcg of reporter DNA, 5 xcexcg of pCH110 DNA, 5 xcexcg of pTZ18U, and 20 xcexcg of DNA/1 mL of transfection solution. The DNA (20 xcexcg) is dissolved in 500 xcexcL of 250 mm sterile CaCl2 which is then slowly (dropwise) added to 500 xcexcL of 2xc3x97HeBS (0.28 M NaCl, 50 mM HEPES, 1.5 mm Na2HPO4, pH 7.05) and incubated at room temperature for 20 min. 20 xcexcL of this mixture is added to each well of cells and remains on the cells for 16 h. At the end of this incubation the precipitate is removed, the cells are washed with media, fresh treatment media is replaced and the cells are treated with either vehicle, 1 nM 17B-estradiol, 1 xcexcM compound or 1 xcexcM compound+1 nM 17B-estradiol. Each treatment condition is performed on 8 wells (n=8) which are incubated for 24 h prior to luciferase assay.
Luciferase Assay: After 24 h exposure to compounds, the media is removed and each well is washed 2xc3x97with 125 xcexcL of PBS lacking Mg++ and Ca++. After removing the PBS, 25 xcexcL of Promega lysis buffer is added to each well and allowed to stand at room temperature for 15 min, followed by 15 min at xe2x88x9280 deg.C and 15 min at 37 degC. 20 xcexcL of lysate is tranferred to an opaque 96-well plate for luciferase activity evaluation and the remaining lysate (5 xcexcL) is used for B-galactosidase activity evaluation (normalize transfection). The luciferan substrate (Promega) added is 100 xcexcL aliquots to each well automatically by the luminometer and the light produced (relative light units) is read 10 seconds after addition. The data is logged and automatically sent to a JMP statistical program for analysis. A hard copy printout is also produced at the time of the assay.
B-galactosidase Assay: To the remaining 5 xcexcL of lysate 45 xcexcL of PBS is added. 50 xcexcl of Promega B-galactosidase 2xc3x97assay buffer is added, mixed well and incubated at 37 deg.C for 1 h. A plate containing a standard curve (0.1 to 1.5 milliunits in triplicate) is set up for each experimental run. The plates are analyzed on a Molecular Devices spectrophotometric plate reader at 410 nm. The optical densities for the unknowns are converted to milliunits of activity by mathematical extrapolation from the standard curve. Analysis of Results: The luciferase data is generated as relative light units (RLUs) accumulated during a 10 second measurement and is automatically transferred to a JMP (SAS Inc) file where background RLUs are subtracted. The B-galactosidase values are automatically imported into the file and these values are divided into the RLUs to normalize the data. The mean and standard deviation is determined from a n=8 for each treatment. Compound activity is compared to 17B-estradiol for each plate. Percentage of activity as compared to 17B-estradiol is calculated as follows:
%=((Estradiol value-control value)/(compound value))xc3x97100
The results of this assay on representative compounds of this invention are shown in Table I.
Reference Compounds: Various reference compounds (1 xcexcM) were assessed for estrogenic and/or antiestrogenic activity (1 xcexcM compound+1 xcexcM 17B-estradiol) by assaying for luciferase activity and corresponding % values compared to 1 nM 17B-estradiol (set to 100%) were calculated. Note there are three orders of magnitude difference in the dose of reference compounds versus 17B-estradiol concentration:
At 1 xcexcM dosages the estriol and estrone would be expected to be about 40% as potent as 17B-estradiol in this assay. The lack of independent activity and antiestrogenic activity of tamoxifen and raloxifene was as predicted as consistent with reports in the literature relating to their effects in a rat uterotrophic assay.
Reference: Tzukerman, M. T., Esty, A., Santiso-Mere, D., Danielian, P., Parker, M. G., Stein, R. B., Pike, J. W. and McDonnell, D. P. Human estrogen receptor transactivatiureal capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Molecular Endocrinology 1994, 8, 21-30.
Specific procedures are described in the following experimental examples. These examples are given to illustrate the invention and should not be construed as limiting the invention set forth in the appended claims.